Phase I resulted in our discovery of low nanomolar level inhibitors of ADP-induced human platelet aggregation, with a unique mechanism of action, that warrant further development as first-in-class antithrombotic drugs. We developed a novel, efficient, high yield method to prepare a series of base and phosphate modified derivatives of diadenosine tetraphosphate (Ap4A). The proposed modifications revealed clear structure-activity trends, and led to the synthesis of highly potent inhibitors of human platelet aggregation that are also stable in plasma. We investigated for the first time the mechanism of action of this class at the level of the three platelet purinoreceptors (P2X1,P2Y1 and P2Y12), and showed that Ap4A and its analogs antagonize both P2Y1 and P2Y12. Moreover this unprecedented simultaneous inhibition of both platelet ADP dependent platelet receptors by a single ligand appears to be highly synergistic - IC50'sfor inhibition of ADP-induced platelet aggregation for the most active compounds is one to two order of magnitude lower than the corresponding IC50'sof inhibition of P2Y1 and P2Y12. Thus these compounds represent a new class of antiplatelet, and, ultimately, antithrombotic drugs with a novel mechanism of action. The specific aims are to 1, expand and tune the structure:activity relationship for inhibiting human platelet aggregation by synthesis and study of six additional Ap4A analogs, 2, evaluate the selectivity/specificity of the new class of Ap4A derivatives, by studying and quantifying the ability of 3-6 most active compounds to interact with non-platelet P2 receptors, utilizing tissue and recombinant receptor models, 3, optimize and scale up the chemical process of synthesis and purification of the class;4, determine the basic pharmacokinetic parameters and identify metabolites of the 3 lead compounds candidates after IV administration in rats, and 5, study the aggregation properties of platelets after IV infusion of these candidates in catheterized rats, and 6, confirm the antithrombotic activity of the selected lead compound in the well-established Folts'canine thrombosis model. A single lead preclinical compound (plus backup) will be designated following these studies. Platelets play critical roles in hemostasis and its pathophysyology. Undesired platelet activation is a result of many common pathologies, e.g. hypertension and arteriosclerosis, and leads to excessive platelet aggregation and the generation of occlusive thrombi (thrombosis). The ischemic events that follow, such as myocardial infarction and stroke, are leading causes of death in the developed world, and antiplatelet drugs have been a major focus of drug development. Aspirin and clopidrogel (Plavix(R), $5.9B sales in 2005) are the most popular of the class today. Because of the vast interpatient variability in response to clopidogrel and newer single-receptor targeted drugs under clinical development, and because of the increased bleeding and morbidity in patients receiving clopidogrel, there is a need for the development of fast, direct acting, and rapidly cleared platelet ADP-receptor antagonists. The overall goal of this research is to identify a novel lead antithrombotic compound for human use, which can be licensed to a major pharmaceutical company for further pre-clinical and clinical development, or alternatively can be developed into a drug candidate by us after securing appropriate partnership or funding. PUBLIC HEALTH RELEVANCE: This project will result in an effective antithrombotic drug that will be used to treat arterial thrombosis in the acute setting. The candidate drug will directly and reversibly inhibit two important receptors involved in platelet aggregation, and will not have the drawbacks of slow and variable action of current drugs such as clopidogrel. The new drug will complement related drugs under development for arterial thrombosis.